The central hypothesis of Project 1 is that a combination of umbilical cord blood (UCB)-derived T regulatory (Treg) cells and thymic progenitors (Tprog) will optimize the safety of UCB by eliminating the risk of severe acute GVHD and enhancing immune recovery. During the current funding period, we initiated the first-inhuman clinical trials with UCB Treg after having developed an expansion culture methodology that routinely yielded Treg that were highly suppressive both in vitro and in vivo using a xenogenic GVHD murine model. During this period, we also evaluated the effect of commonly used immunosuppressive agents on Treg at concentrations routinely achievable in transplant recipients. On the basis of these results, we initiated a second clinical trial with UCB-Tregs using rapamycin (Rapa) rather than Cyclosporin A in combination with mycophenolate mofetil (MMF) to optimize in vivo expansion and half life as well as potency. Over the next grant period, our goal is to develop an integrated, cell-based approach that will ultimately reduce the risk of acute GVHD, permit a reduction in the need for prolonged posttransplant immunosuppression, and enhance the pace of immune reconstitution. To accomplish these goals, we will first establish the maximum tolerable dose of Treg using newly developed improved methods for large scale Treg manufacture, and then demonstrate the potency of partially HLA matched Treg in prevention and off the shelf HLA unmatched Treg in treatment of acute GVHD. We will also test novel approaches to T cell immune reconstitution by determining the optimal balance of Treg and T effector cells in the UCB graft without posttransplant GVHD prophylaxis, and subsequently adding Tprog to this treatment platform to enhance thymopoiesis and T cell immune reconstitution. At the conclusion of these studies, we will have demonstrated the safety profile and potency of UCB Treg and established a new treatment paradigm without pharmacologic immunosuppression for evaluating safety and efficacy of Tprog.